In both plants and animals, a large number of unique cell types must be specified during development. It has become increasingly clear that as cells differentiate, not only must genes be transcriptionally activated, but the transcription of others must be repressed. In animals, mutations in genes that regulate transcriptional repression can lead to many types of cancer or result in embryonic lethality. Therefore it is important to identify and understand the mechanisms by which these proteins act. We have identified two proteins in Arabidopsis, TOPLESS (TPL) and HISTONE DEACETYLASE 19 (HDA19), that work in concert to repress transcription during embryogenesis and flower development. TPL encodes a transcriptional corepressor that forms a complex with HDA19 to maintain cell fates during development. Unlike in animal systems where mutations in similar proteins are embryonic lethal, mutations in both TPL and HDA19 are completely viable in Arabidopsis, allowing us to study their roles at multiple developmental stages. In AIM1, we will perform chromatin immunoprecipitations followed by massively parallel sequencing (ChIP seq) to identify the common direct targets of TPL and HDA19 during embryogenesis. We will then employ mis-expression and loss-of-function analysis to determine the function of these downstream targets. In AIM2, we will perform ChIP seq on TPL and HDA19 during floral development to elucidate their downstream targets at a second stage of development. We will then analyze the extent of histone acetylation on a subset of target promoters over time, as well as perform expression, mis-expression and loss-of-function studies. In AIM3 we will perform genetic screens on hda19 mutants to identify other proteins in the TPL/HDA19 pathway. These experiments will allow us to explore one of the mechanisms that plants use to specify and maintain cell types during development. It will allow us to compare the proteins and logic used in plants to those used in similar processes in animal development, leading to a better understanding of both. PUBLIC HEALTH RELEVANCE: Understanding how different cell types are specified in an organism is fundamental to public health as this process is critical for development, and disruption of it leads to various diseases in humans. This proposal aims to explore one aspect of this process, transcriptional repression, which has been linked to several human pathologies such as cancer. The findings from this study will aid in our understanding of the mechanisms that govern this important process.